We propose to continue our efforts to identify the specific electron carriers of the respiratory chain that are responsible for translocating electric charges and H ion across the mitochondrial membrane, thus generating an energy-rich electrochemical gradient which is used in the synthesis of ATP. In particular, we shall concentrate on energy-conserving site 3, that promoted by cytochrome oxidase, since our investigations indicate that this is the key enzyme in the respiratory chain responsible for regulating the overall rate of H ion extrusion and oxidative phosphorylation. We also propose to examine the transport mechanisms in the inner membrane that bring about H ion translocation, in order to determine whether the primary gradient employed in oxidative phosphorylation is formed between two bulk phases or whether it is generated within or on the inner membrane itself. We also propose to examine the membrane transport menchanisms for ATP, ADP and phosphate that participate during oxidative phosphorylation and during ATP-dependent reverse electron flow, which is an important regulatory process in the coordination of glycolysis and respiration. We also propose to investigate the regulation of Ca2 ion influx and efflux by respiring mitochondria and how Ca2 ion homeostasis serves as a signaling agent in the bio-energetic relationships between the cell cytosol and the mitochondria.